Epstein-Barr virus (EBV), a human gammaherpesvirus, is largely B lymphotropic and possesses a unique set of latent cycle genes whose coordinate expression can drive the proliferation of latently infected B cells. This process can be studied in vitro, where experimental infection of resting B cells leads to the outgrowth of virus-transformed lymphoblastoid cell lines (LCLs) expressing the full range of EBV latent proteins; these include the nuclear antigens EBNA1, -2, -3A, -3B, -3C, and -LP and the latent membrane proteins LMP1, -2A, and -2B (25, 42). Immediately postinfection, viral transcription initiates from the latent cycle promoter Wp. The long primary transcripts thus produced are capable of generating all six EBNA mRNAs but appear to be preferentially, though not exclusively, processed to mRNAs encoding the EBNA2 and EBNA-LP proteins (2,13,62,63). Their appearance leads to activation of an alternative upstream promoter, Cp, from which the full complement of EBNA proteins is expressed, as well as to activation of the more distant LMP promoters (1, 54, 60, 61, 67), thereby completing the full range of latent protein expression. The activation of Cp is followed by a gradual waning of Wp transcription such that Cp is dominant over Wp in most established LCLs (4, 63).Virus-driven B-cell growth transformation is also observed in vivo during primary EBV infection. Thus, Wp, Cp, and LMP transcripts are detectable in the circulating B-cell pool of infectious mononucleosis (IM) patients (58). However, after resolution of the acute infection and establishment of a lifelong virus carrier state, virus-infected cells in the blood are only detectable within the resting memory B-cell pool (5, 32) and show a different program of viral transcription; in particular, Wp, Cp, and most if not all of the LMP promoters (with the possible exception of LMP2A) appear to be silent (41, 58). The mechanisms of promoter regulation which effect this switch to a more restricted form of virus latency are still not understood. However, some interesting potential clues have come from the study of EBV genome-positive malignancies, such as Burkitt's lymphoma (BL) and nasopharyngeal carcinoma, which also show more limited patterns of latent protein expression than do LCLs (42). In particular, Wp and Cp are silent in such tumors, and the only detectable EBNA, EBNA1, is expressed from a downstream EBNA1-specific promoter, Qp (48-50). Digestion of viral DNA from these tumors, using restriction enzymes that are either sensitive or insensitive to the presence of a methylated cytosine in the target sequence, indicated that several regions of the genome were more heavily methylated in tumor cells than in LCLs (3,4,11,20,22,31,51). These regions included the BamHI W and C fragments, within which Wp and Cp are situated, but not the region around Qp. Given the increasing evidence for methylation at CpG dinucleotides as a general mechanism of promoter silencing in eukaryotic cells (10), this raised the possibility that methylation of EBV latent promoters...

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Epstein-Barr virus (EBV)-Epstein-Barr virus (EBV) is the best known of the ␥1 herpesviruses, a group of closely related B-lymphotropic agents of primates which have evolved a unique strategy through which to access, disseminate, and persist within the B-lymphoid system. The essential features of that strategy are threefold. First, EBV can efficiently access target B lymphocytes through an interaction between the major viral envelope glycoprotein gp340 and the complement receptor

Epstein±Barr virus, a human gammaherpesvirus, possesses a unique set of latent genes whose constitutive expression in B cells leads to cell growth transformation. The initiation of this growth transforming infection depends on a viral promoter in BamHI W (Wp) whose regulation is poorly understood. Using Wp reporter constructs in in vitro transfection assays, we found that Wp was 11-to 190-fold more active in B cell than in non-B cell lines and that three regions of the promoter (termed UAS1, UAS2, and UAS3) contributed to transcriptional activation. The upstream regions UAS3 (Ϫ1168 to Ϫ440) and UAS2 (Ϫ352 to Ϫ264) both functioned in a cell lineage-independent manner and were together responsible for the bulk of Wp activity in non-B cells; mutational analysis indicated the importance of a YY1 binding site in UAS2 in that context. By contrast, UAS1 (Ϫ140 to Ϫ87) was B cell specific and was the key determinant of the promoter's increased activity in B cell lines. Mutational analysis of UAS1 sequences combined with in vitro bandshift assays revealed the presence of three binding sites for cellular factors in this region. When mutations that abolished factor binding in bandshift assays were introduced into a Wp reporter construct, the loss of any one of the three UAS1 binding sites was sufficient to reduce promoter activity by 10to 30-fold in B cells. From sequence analysis, two of these appear to be novel transcription factor binding sites, whereas the third was identified as a cyclic AMP response element (CRE). Our data indicate that this CRE interacts with CREB and ATF1 proteins present in B cell nuclear extracts and that this interaction is important for Wp activity.

The programme of Epstein-Barr virus (EBV) gene expression that leads to virus-induced growth transformation of resting B lymphocytes is initiated through activation of the BamHI W promoter, Wp. The factors regulating Wp, and the basis of its preferential activity in B cells, remain poorly understood. Previous work has identified a B cell-specific enhancer region which is critical for Wp function and which contains three binding sites for cellular factors. Here we focus on one of these sites and show, using bandshift assays, that it interacts with three members of the CREB/ATF family of cell transcription factors, CREB1, ATF1 and ATFa. A mutation which abrogates the binding of these factors reduces Wp reporter activity specifically in B cell lines, whereas a mutation which converts the site to a consensus CREB-binding sequence maintains wild-type promoter function. Furthermore Wp activity in B cell, but not in non-B cell, lines could be inhibited by cotransfection of expression plasmids expressing dominant negative forms of CREB1 and ATF1. Increasing the basal activity of CREB/ATF proteins in cells by treatment with protein kinase A or protein kinase C agonists led to small increases in Wp activity in B cell lines, but did not restore promoter activity in non-B cell lines up to B cell levels. We conclude that CREB/ATF factors are important activators of Wp in a B cell environment but require additional B cell-specific factors in order to mediate their effects. IntroductionEpstein-Barr virus (EBV), a lymphotropic herpesvirus with potent B cell-transforming properties, is aetiologically linked to a number of human malignancies and is the causative agent of infectious mononucleosis, a self-limiting lymphoproliferative disease (Rickinson & Kieff, 1996). The experimental infection of resting B cells in vitro leads to the outgrowth of EBV-transformed lymphoblastoid cell lines (LCLs). Such LCLs proliferate indefinitely in culture as the result of the coordinate action of a limited set of viral gene products (Kieff, 1996) ; these include the six nuclear antigens (EBNAs 1, 2, 3A, 3B, 3C and LP), three latent membrane proteins (LMP1, LMP2A and LMP2B), products of the highly spliced BamHI A transcripts and two small non-polyadenylated RNAs (EBERs 1 and 2). The initiating event in this growth transformation pathway is the activation of a virus promoter, Wp, located in the BamHI W repeat region of the EBV genome (Sample et al., 1986 ;Woisetschlaeger et al., 1990 ;Alfieri et al., 1991). This promoter drives the initial expression of EBNA2, a Author for correspondence : Andrew Bell.Fax j44 121 414 4486. e-mail a.i.bell!bham.ac.uk transactivator which, together with a second protein, EBNA-LP, is responsible for the induction of the remaining latent genes (Abbot et al., 1990 ;Fa/ hraeus et al., 1990 ;Wang et al., 1990 ;Sung et al., 1991 ; Woisetschlaeger et al., 1991 ; ZimberStrobl et al., 1991 ;Jin & Speck, 1992 ; Rooney et al., 1992 ;Harada & Kieff, 1997 ;Nitsche et al., 1997).Although there is increasing understanding of the m...